The goals of this project are to identify highly oxidized proteins in the cell, understand their physiologic function, and identify changes in structure and function during aging. Using an immunological method for the detection of oxidized proteins, we were able to identify a highly modified protein in rat liver. The protein was purified and characterized as carbonic anhydrase, isoenzyme III (CAIII), an enzyme which catalyzes hydration of carbon dioxide. Since it is well known that an increased carbonyl content is a marker of oxidative stress and aging, we purified CAIII from 2, 10, and 18 month old rats and the proteins were characterized. All three preparations were highly oxidatively modified as assessed by their carbonyl content. CAIII has been found by others to be extensively glutathiolated, a reversible modification thought to be important during oxidative stress. Glutathiolation almost doubled during aging. While the physiological role of CAIII is unknown, these results suggest that it functions in an oxidizing environment, which leads to its oxidative modification. The enzyme has three known catalytic activities, and the specific activities for carbon dioxide hydration and for ester hydrolysis decreased during aging by a modest 30%. However, the third activity, that of a phosphatase, was virtually lost during aging. The sequence CxxxxxR has been described by Zhang et al. as a motif for the phosphatase activity and inspection of the sequence of CAIII demonstrated the presence of this motif (C183-LFPAC-R189). Investigation of the effect of glutathiolation in the CAIII's phosphatase activity revealed that glutathiolation of C188 doubled the phosphatase activity while removal virtually eliminated activity. The loss of activity is reversible, as re- glutathiolation restored activity. We hypothesize that glutathiolation acts as a regulatory mechanism for the phosphatase activity of CAIII.